JP3754648B2 - Production of barley with reduced gel protein - Google Patents

Description

Technical field
The present invention relates to producing barley with reduced gel protein in barley seeds.
Background art
Barley (Hordeum vulgare) seeds contain a large amount of seed-specific protein (seed storage protein), of which 35-55% is alcohol-soluble hordein (Shewry 1993, Barley: Chemistry and Technology.pp164: American Association of Cereal Chemists).
This hordein is classified into four types, B, C, D, and γ, based on its locus and amino acid composition, and the content ratio to all hordeins is 70 to 80% for B, 10 to 20% for C, and 5 for D. % Or less.
In addition to having a very important role as an amino acid source for yeast in beer brewing, these hordeins have been reported to affect beer chromaticity and the formation of cold turbidity. These reports examine the effects of hordein as a whole on beer quality and beer production processes, not individual hordeins.
For example, Baxter (Baxter, 1980, Brewers Digest 55: 45-47) found that adding a reducing agent to mash improves the filtration rate of wort. Furthermore, von den Berg et al. (Van den Berg et al. 1981, Proceeding of the EBC congress, Copenhagen, 47: 461-469) found that a gel-like protein precipitated by protein extraction from barley with 1.5% SDS and then centrifuged. Aggregates, ie gel proteins, have been reported to adversely affect wort filterability. These reports suggest a relationship between gel protein in which B and D hordeins are polymerized by disulfide bonds and wort filterability.
It has also been reported that there is a strong negative correlation between this gel protein and malt extract (Smith and Lister (1983), Journal of Cereal Science 1: 229-239, Smith and Simpson (1983), Journal of Cereal Science 1: 185-197, Skerritt and Janes (1992), Journal of Cereal Science 16: 219-235, Howard et al. (1996), Journal of Cereal Science 24: 47-53).
In addition, Brennan (Brennan et al. (1998), Journal of Cereal Science 28: 291-299) examined the presence of D hordein and the amount of gel protein using isogenic lines using barley lines lacking D hordein. , Suggesting a relationship between the two.
From such a viewpoint, it has been suggested that if gel protein can be reduced by some method, wort filterability and wort extract yield can be improved.
Disclosure of the invention
However, as a method for reducing the gel protein so far, it is possible to use a barley line having a low gel protein content as a single parent (Brennan et al. (1998), Journal of Cereal Science 28: 291-299) or irradiation, etc. However, none of these methods were efficient.
That is, in the method using the above-mentioned crossing, considerable time, labor, breeding field, and appropriate breeding techniques are required to obtain a variety or line having a reduced target gel protein. In addition, many systems must be tested for selection. Therefore, with this method, it has been extremely difficult to produce barley with reduced gel protein economically and efficiently.
Another method for inducing mutants using radiation or the like is that the frequency of mutation is low, and the position where the mutation is inserted is random. To obtain it requires a considerable scale of mutagenesis testing. In addition, there is a problem that a special facility is required to perform such radiation irradiation, and the place where the production operation is performed is restricted.
On the other hand, due to recent advances in molecular biology, methods for modifying plants using genetic engineering techniques have been developed, and by development and improvement of barley transformation techniques such as protoplast method, particle bombardment method, and Agrobacterium method. Transformed barley can be obtained relatively easily and in a short period of time.
Thus, the present inventors have conducted extensive research to solve these problems, and as a result, have developed a method for producing barley in which gel proteins are efficiently and economically reduced using transformation techniques.
The present invention is intended to reduce the amount of gel protein in barley by suppressing the production of D-type contained in trace amounts in hordein, that is, D hordein. In the present invention, D hordein accounts for only about 5% of all hordeins, but it is possible to reduce the amount of gel protein more than expected by suppressing the production of D hordein. By what they found.
That is, the present invention is a method for producing barley with reduced gel protein that can be aggregated in the form of a gel during protein extraction from barley, and is capable of suppressing the production of endogenous D hordein protein in barley. Is introduced into barley, and the introduction of the D hordein expression-suppressing nucleic acid suppresses the production of the D hordein protein, thereby reducing the amount of gel protein.
According to the said invention, the D hordein expression suppression nucleic acid is introduce | transduced into a barley, the production of the said D hordein protein is suppressed, and it becomes possible to reduce a D hordein protein fraction from gel protein. Furthermore, although this D hordein protein constitutes only a part of the gel protein, the reduction of the D hordein protein occupying this part of the protein eliminates other proteins constituting the gel protein from the gel protein. Or it becomes possible to reduce. As a result, according to the present invention, the total gel protein amount can be reduced more than expected.
The present invention is characterized in that, in the above-mentioned invention, the D hordein expression-suppressing nucleic acid can express an antisense RNA competing with an endogenous D hordein RNA of barley.
According to the above invention, antisense D hordein RNA is expressed in the barley, and this is combined with the endogenous D hordein RNA, so that translation into the protein is inhibited and production of D hordein protein can be suppressed. .
The present invention relates to a D hordein expression-suppressing nucleic acid that generates an antisense RNA that can be complementary to an endogenous D hordein RNA generated in a barley, wherein the antisense RNA is generated downstream of a promoter that operates in the barley. As described above, the D hordein coding sequence encoding D hordein is ligated in the reverse direction.
According to the D hordein expression-suppressing nucleic acid of the present invention, antisense D hordein RNA is transcribed from a promoter, and this antisense D hordein RNA is complementarily bound to the endogenous D hordein RNA and is based on the D hordein RNA. Translation of D-hordein protein can be inhibited.
Further, in the D hordein expression-suppressing nucleic acid of the present invention, the D hordein coding sequence is (1) a base sequence described in SEQ ID NO: 1, (2) a base sequence described in SEQ ID NO: 2, and (3) SEQ ID NO: 1. A nucleotide sequence encoding an RNA having at least one of substitution, deletion, addition, or insertion in the described nucleotide sequence, and complementary to the endogenous D hordein RNA of the barley, or (4) described in SEQ ID NO: 2 Wherein the base sequence encodes an RNA having at least one of substitution, deletion, addition, and insertion and capable of complementing the endogenous D hordein RNA of the barley.
According to these base sequences, when they are expressed, they can be complementarily bound to the endogenous D hordein RNA, and the production of D hordein protein can be suppressed.
The present invention also relates to a vector comprising the D hordein expression-suppressing nucleic acid.
Thus, by connecting the expression-suppressing nucleic acid to the vector, the expression cassette can be stably held, and the expression cassette can be freely amplified.
The present invention is characterized in that a selectable marker expression cassette capable of expressing a selectable marker is provided in the vicinity of the D hordein expression-suppressing nucleic acid.
According to the above invention, by providing a selection marker expression cassette, it is possible to select a strain into which the D hordein expression-suppressing nucleic acid has been introduced, using the selection marker as a clue, and a strain in which the expression of D hordein is suppressed. That is, it becomes possible to rapidly identify a gel protein-reduced strain.
The gel protein-reducing barley production kit of the present invention comprises the D-hordein expression-suppressing nucleic acid or the vector, and introduces the D-hordein expression-suppressing nucleic acid or the vector into barley to reduce the barley gel protein. .
According to the present invention, since the D hordein expression-suppressing nucleic acid or the above-mentioned vector is included, it is possible to obtain a barley with a reduced gel protein simply by introducing these into a barley and performing transformation. .
The present invention also includes a barley produced by any one of the above-described methods for producing a gel protein-reduced barley or the gel protein-reduced barley production kit according to any one of claims 8 to 9.
According to the barley of the present invention, only the amount of D hordein protein, which can be said to be a trace amount of hordein, is reduced, and thereby the amount of gel protein is reduced, so that wort is considered to be related to the amount of gel protein. The improvement of filterability and malt extract yield can be expected.
[Brief description of the drawings]
FIG. 1 is a diagram showing the structure of the D hordein expression suppression vector used in Example 1.
FIG. 2 is a diagram showing a construction method of the D hordein expression suppression vector used in Example 1.
FIG. 3 is a view showing the results of transgene confirmation by Southern blotting of the transformed barley of Example 2.
FIG. 4 is a diagram showing analysis by SDS polyacrylamide electrophoresis of the hordein fraction of the transformed barley seeds of Example 3.
5A and 5B are diagrams showing the results of gel protein quantification of the transformed barley seeds of Example 4. FIG.
FIG. 6 is a graph showing changes in the gel protein composition of the transformed barley seeds of Example 5.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described.
1. D-hordein gene expression suppression method
Suppression of D-hordein expression in barley is because D-hordein protein is processed through the process of transcription from chromosomal gene to RNA and translation from RNA to protein. Can be executed.
For example, examples of a method for suppressing the expression of D hordein protein include an antisense method, a co-supression method, and a ribozyme method. Moreover, not only the method of inhibiting the production | generation process of D hordein protein, but you may suppress the expression by changing D hordein gene area | region used as the foundation of the production | generation of this D hordein protein. As such an expression suppression method, for example, a gene targeting method can be employed.
Since the purpose is to suppress the expression of D hordein, any method described above may be adopted as long as it can suppress the expression of D hordein protein as described above, but it is highly reliable and specific. As a high method, an antisense method can be preferably used.
2. Preparation of nucleic acid for suppressing expression of D-hordein gene
The configuration of the nucleic acid for suppressing the expression of D hordein varies depending on the expression suppression method employed.
(1) Antisense method
In the antisense method, antisense RNA is specifically bound to the target D hordein mRNA, thereby inhibiting translation of D hordein mRNA into protein. Therefore, when the antisense method is employed, antisense D hordein RNA can be used as an expression-suppressing nucleic acid, and expression of D hordein protein can be suppressed by directly supplying this antisense RNA to barley. .
Moreover, as this expression suppression nucleic acid, Preferably, the expression cassette which expresses antisense RNA is employable. By transforming barley with this expression cassette, antisense RNA can be stably supplied in the transformed barley, and this property can be applied to the next generation seed group obtained from this transformant. It can also be inherited.
The antisense D hordein expression cassette can be constructed as follows. The barley D hordein gene is ligated in the reverse direction, operably downstream of the promoter expressed in barley ripening endosperm tissue, and further, a transcription termination factor that functions similarly in the barley ripening endosperm tissue is ligated downstream thereof. Can be created.
Here, the “barley D hordein gene” is ligated in the reverse direction downstream of the promoter so that antisense RNA can be generated. The “barley D hordein gene” introduced into this expression cassette does not need to have the entire region of the D hordein gene, but may have a length that can inhibit translation from the endogenous D hordein mRNA. For example, a partial sequence of the D hordein gene may be used.
Conversely, the “barley D hordein gene” in the cassette may be longer than the endogenous D hordein mRNA, as long as it can similarly inhibit translation based on the endogenous D hordein mRNA. A DNA fragment may be added.
As the barley D hordein gene, specifically, the sequence shown in SEQ ID NO: 1 or 2 can be preferably used, but a sequence substantially identical to these sequences can also be used. This substantially identical sequence is a sequence having at least one of substitution, deletion, addition, and insertion in the above sequence and capable of suppressing the expression of the endogenous D hordein gene.
The transgene in the expression cassette is not limited to the “barley D hordein gene”, and a gene having high homology with the D hordein gene can also be used. For example, a wheat polymer glutenin subunit gene or a rye polymer secalin gene can be used as long as it can complementarily bind to the endogenous D hordein mRNA.
In addition, the transgene in the expression cassette used here may be genomic DNA, cDNA, a gene in which these genes are synthesized, modified, or the like.
The “promoter” in the expression cassette is not particularly limited as long as it has promoter activity in the ripening endosperm tissue of barley. Examples thereof include B hordein promoter, C hordein promoter, γ hordein promoter, β amylase promoter, wheat polymer glutenin subunit promoter, rye polymer secalin promoter, CaMV35S promoter, actin promoter and the like.
The transcription terminator is not particularly limited as long as it functions in the ripening endosperm tissue of barley, and for example, a NOS terminator can be used.
(2) co-supression method
The D-hordein expression-suppressing nucleic acid in the co-supression method is, for example, a barley D hordein gene is linked as a transgene in the forward direction downstream of a promoter expressed in barley ripening endosperm tissue, and further, a transcription such as NOS terminator is further downstream. It can be created by linking termination factors.
The transgene used here is a DNA fragment necessary for reducing D hordein. For example, the D hordein gene can be preferably used. This transgene can then be inserted in the positive direction between the promoter and the transcription terminator. In addition, the transgene does not need to include the entire region expressed as mRNA, and may be a short gene fragment. Conversely, the transgene may be longer than the entire region expressed as mRNA, or may be an arbitrary DNA fragment added thereto.
In addition to the D hordein gene, the transgene can be used as long as it is highly homologous to the D hordein gene. For example, a wheat polymer glutenin subunit gene or a rye polymer secalin gene can be used.
The transgene used here includes genomic DNA, cDNA, and genes obtained by synthesizing, modifying, and manipulating these genes.
The “promoter” may be anything as long as it has promoter activity in the ripening middle endosperm tissue. Examples include B hordein promoter, C hordein promoter, γ hordein promoter, β amylase promoter, wheat polymer glutenin subunit promoter, rye polymer secalin promoter, CaMV35S promoter, actin promoter and the like.
(3) Ribozyme method
The ribozyme method is a method for suppressing the expression of D hordein by degrading D hordein mRNA with a ribozyme (RNA enzyme). Accordingly, the D hordein gene expression-suppressing nucleic acid used in this method is a gene encoding a ribozyme designed so as to be able to degrade the expressed D hordein mRNA, and this gene is converted into a promoter and a transcription terminator as described above. It can comprise by connecting in a positive direction between.
(4) Construction of the nucleic acid for suppressing D hordein expression described above requires operations related to gene cloning, such as restriction enzyme treatment, DNA ligation treatment, and transformation of E. coli, which are performed using conventional techniques. Is done. For example, Molecular Cloning (Sambrook, Fritsch, Maniatis. Cold Springharbor Laboratory) can be referred to.
The specific gene expression-suppressing cassette described in this section can be introduced into a plant chromosome in the form of a linear chain, or can be incorporated into an arbitrary plasmid as described later and used as a specific gene expression-suppressing gene expression vector.
3. Creation of specific gene expression suppression vector
The expression-suppressing nucleic acid can be used directly as a nucleic acid fragment, but can be inserted into a vector and used as an expression-suppressing vector. The expression suppression vector can be prepared by inserting the above expression suppression nucleic acid into an arbitrary plasmid. In addition, an expression suppression vector can be simultaneously prepared together with the nucleic acid for expression suppression by sequentially linking a promoter, a structural gene, and a transcription termination factor to an arbitrary plasmid.
As the plasmid that can be used here, any commercially available plasmid such as plasmid pBI101 may be used, but it is preferable to select it according to the purpose. For example, when it is desired to collect a large amount of vector, it is desirable to select a plasmid having a large copy number. Furthermore, the plasmid can be provided with a selection marker based on a drug (neomycin or the like) or a nutritional component (for example, amino acid requirement) or the like that serves as an index for introducing the expression vector into an organism or the like. By providing this selection marker, a plant introduced with the expression vector can be selected using the selection marker as an index.
In order to construct the above-described D hordein expression suppression vector, operations related to gene cloning such as restriction enzyme treatment, DNA ligation treatment, and transformation of Escherichia coli are necessary, and these can be performed using conventional techniques. it can. In this case, for example, Molecular Cloning (Sambrook, Fritsch, Maniatis, Cold Springharbor Laboratory) can be referred to.
4). Introduction of expression-suppressing nucleic acid or expression-suppressing vector containing the same into plants
As a cell into which an expression-suppressing nucleic acid or an expression-suppressing vector containing the same is introduced, a plant cell having redifferentiation ability is preferable. Examples of such cells include immature embryo-derived cells and pupa-derived cells.
As a method for introducing a nucleic acid, a known method can be used. Specifically, in addition to the polyethylene glycol method, an electroporation method, a particle gun method, an Agrobacterium method, and the like can be given.
5. Transformed plant
Unlike the pre-transformation properties, a plant into which an expression-suppressing nucleic acid or an expression-suppressing vector has been introduced as a foreign DNA as described above differs from the properties before transformation in that D hordein is not produced or the amount produced is reduced. Form new varieties or strains with them.
Moreover, plants having the same properties, such as reduced D-hordein, can also be obtained by crossing the transformed plants prepared by the above expression suppression method with other varieties and strains. This essentially corresponds to a transformed plant prepared by the expression suppression method.
6). Quantification and qualification of gel proteins
A known method can be used for extracting and quantifying gel protein from seeds grown in transformed barley. For example, using the method of Smith and Lister (Smith and Lister (1983), Journal of Cereal Science 1: 229-239) or Skerritt and Janes (Skerritt and Janes (1992), Journal of Cereal Science 16: 219-235) Can do. SDS polyacrylamide electrophoresis is effective for examining the composition of the obtained gel protein, but it is also possible to use high performance liquid chromatography. B, C, and D hordeins can be identified based on information known from the molecular weight. For example, Shewry (Shewry (1993), Barley: Chemistry and Technology. Pp164: American Association of Cereal Chemists) is helpful.
[Example]
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[Example 1] Preparation of D-hordein expression suppression vector
The structure of an expression suppression vector that expresses antisense RNA against D hordein mRNA is shown in FIG.
As shown in FIG. 1, the D hordein expression suppression vector is provided with an antisense expression cassette 10 that expresses antisense RNA against D hordein mRNA as a D hordein expression suppression nucleic acid. In this antisense expression cassette 10, downstream of the D hordein promoter (D hordein P) 14 (SEQ ID NO: 3), D hordein cDNA 16 (SEQ ID NO: 1 or 2) is connected in the reverse direction, that is, in the 3 ′ → 5 ′ direction. ing. Further, a NOS terminator 18 is connected downstream of the reverse D hordein cDNA 16.
In addition, the D hordein expression suppression vector is provided with a selection marker expression cassette 12 in a form head-to-head with the antisense expression cassette 10, and the selection marker expression cassette 12 includes an arf promoter 20. A neomycin phosphotransferase (NPTII) gene 22 is connected downstream, and a NOS terminator 24 is connected downstream thereof.
The construction of this D hordein vector is shown in FIG.
A vector carrying the DPP3 promoter, which is the D hordein promoter 14, was prepared, and a DPP3Nhe2 vector in which the HindIII site of the multicloning site of this vector was changed to the NheI site was prepared. This DPP3Nhe2 vector was digested with restriction enzymes BamHI and EcoRI, a BamHI-EcoRI fragment containing NOS terminator 18 derived from pBI221 (manufactured by Clonetec) as a transcription terminator was inserted therein, ligated and ligated, and DPP3NheT A vector was obtained (S01).
Next, this DPP3NheT vector was digested with the restriction enzyme BstX1, the ends were blunted and reconnected to obtain a DPP3NheTBXD vector (S02). The DPP3NheTBXD vector obtained here has a PmaCI site and a SacI site between the D hordein promoter and the NOS terminator.
Next, the DPP3NheTBXD vector was digested with SacI to prepare a blunted end (S03). On the other hand, pDH4 or pDH6 carrying D hordein cDNA 16 is digested with EcoRI, and a blunt-ended fragment of EcoRI fragment containing the entire region of D hordein cDNA (derived from pDH4: SEQ ID NO: 1, pDH6: SEQ ID NO: 2) is prepared. (S04). These two fragments were ligated, and D hordein cDNA 16 was selected in the direction opposite to that of promoter 14 to be DPADH4S and DPADH6S, respectively (S05).
As described above, in order to introduce the selection marker expression cassette 12 into the DPADH4S and DPADH6S vectors in which the antisense expression cassette 10 was formed, the selection marker expression cassette was adjusted from pSBG121NH.
To this pSBG121NH, a neomycin phosphotransferase gene (manufactured by Pharmacia, hereinafter nptII gene) is linked downstream of the arf promoter (SEQ ID NO: 4), and a NOS terminator is further linked downstream thereof. Therefore, this pSBG121NH was digested with NheI, an expression cassette containing the NPTII gene 22 was recovered (S06), and this fragment was introduced into the NheI sites of the above-mentioned DPADH4S and DPADH6S. After the introduction, selection marker expression cassette 12 and antisense expression cassette 10 linked head-to-head were selected and designated as DPADH4NPT and DPADH6NPT (S07).
[Example 2] Transformation of barley
1. Barley transformation
Barley transformation was performed by introducing a D hordein expression suppression vector (DPADH4NPT or DPADH6NPT) into protoplasts obtained from cell lines derived from immature embryos of barley variety Igri using the polyethylene glycol method.
Specifically, the two types of vectors prepared in Example 1 were purified using a Qiagen column (manufactured by Quiagen), respectively, and then TE buffer (10 mM Tris HCl (pH 7. 5) was adjusted to a concentration of 1 μg / μl. 5), suspended in 1 mM EDTA). On the other hand, protoplasts were prepared by enzyme treatment and purification from a liquid suspension culture cell line of cultivar Igri.
Vector introduction into this protoplast was basically performed according to the method of Funatsuki et al. (Funatsuki et al. (1995) Theoritical and Applied Genetics 91: 707-712). That is, the obtained protoplast was suspended in 250 μl of Ca-S (calcium sorbitol solution) containing 50 μg of the above vector, 100 mM CaCl 2, 0.6 M sorbitol, 0.1% (w / v) MES and adjusted to pH 5.7. It became cloudy.
To this suspension, 600 μl of Ca-S PEG (pH 7.0) containing 40% (w / v) PEG (polyethylene glycol, molecular weight 1540) was added dropwise, shaken several times, and allowed to stand for 5-10 minutes. . After diluting by adding 10 ml of LW solution, the protoplast was recovered by centrifugation. This protoplast was suspended in 1 ml of modified L1 medium (Lazzeri et al. 1991) containing 1.8% Sea Plaque Agarose (manufactured by FMC) and 0.4 M maltose, and cultured with nurse cells.
2. Selection of vector-introduced colonies and growth on plants
After 2-3 weeks from the start of protoplast culture, the liquid medium and nurse cells were removed. Furthermore, in order to select only colonies having the NPTII gene as primary selection, the obtained colonies were cultured in a liquid medium containing 20 or 25 μg / ml of G418 (Geneticin, manufactured by Gibco).
The G418-resistant colonies were subjected to shaking culture for about 14 days, and then transferred to a redifferentiation medium containing G418 at the same concentration as the primary selection as a secondary selection. Colonies that show callus and embryoid body formation or shoot differentiation during the secondary selection process are transplanted into a modified L3 medium containing no G418 as transformed cells derived from independent protoplasts. Promoted redifferentiation. When the green shoot reached 1-2 cm, the petri dish was moved under strong light and cultured, then transplanted to a medium without hormones to promote rooting and potted.
As a result of the above transformation experiments, 18 lines and 66 individuals were obtained from the group that attempted to introduce DPADH4NPT, and 12 lines and 29 individual regenerated plants were obtained from the group that attempted to introduce DPADH6NPT. . In addition, all the barley tested were grown in an artificial weather chamber controlled at 16 ° C., 16 hours long, and 20000-30000 looks illumination.
3. Confirmation of the presence of vectors in plants
Whether or not the introduced D hordein expression suppression vector (DPADH4NPT or DPADH6NPT) was present in the regenerated plant body was confirmed using the Southern hybridization method.
First, 1 g of regenerated plant leaves were prepared, ground in liquid nitrogen, and then extracted with CTAB method (Murray et al. 1980, Nucleic Acids Res. 8: 432 1-4325). The obtained DNA was digested with HincII, subjected to agarose gel electrophoresis, and after alkali denaturation, was capillary blotted onto a nylon membrane (Boehringer). The probe used was a vector in which the insert of pDH4 was ligated in the opposite direction, and a label labeled with DIG-high prime (manufactured by Boehringer) using a fragment of about 1.7 Kb obtained by digesting pDH4R6 with HincII as a template. .
Southern hybridization was performed according to the Boehringer manual. The membrane was washed after hybridization with 2 × SSC, 1% SDS for 30 minutes at room temperature, and then with 0.1 × SSC, 0.1% SDS at 42 ° C. for 30 minutes twice.
The signal detection method followed the Boehringer manual. As a result of detecting the transgene for each line of the regenerated plant obtained by Southern hybridization, 12 lines, 48 individuals, among the redifferentiated plants obtained from the section where the introduction of the D hordein expression suppression vector was attempted, Transgenes were detected from 23 strains of 10 lines among the redifferentiated plants obtained from the area where DPADH6NPT was introduced.
However, here, when a transgene was detected in the representative of each line, it was calculated that all individuals belonging to that line had the transgene. FIG. 3 shows an example of transgene detection by Southern hybridization. As is clear from this, the signal of the endogenous D hordein gene is only detected in the DNA obtained from the redifferentiated plant not introduced with the gene used as a control (lane 8 in FIG. 3). From the differentiated plant, it was confirmed that signals other than the endogenous gene, that is, a transgene was present (FIG. 3, lane 1 to lane 7, lane 9 to lane 12).
[Example 3] Confirmation of effect of D-hordein expression suppression vector
The effect of the D hordein expression suppression vector was confirmed by measuring the D hordein content in the seed of the transformant from the band intensity of electrophoresis as shown below.
1. Extraction of protein fraction from seed
In the above, proteins were extracted from seeds (hereinafter referred to as T1 seeds) that were cultivated in transformants that were confirmed to have introduced the D hordein expression suppression vector. In this extraction, one seed was pulverized finely using a hammer, USD buffer (8 M urea, 0.1 M dithiothreitol, 50.0 mM sodium carbonate) was added and stirred, and then incubated at 60 ° C. for 30 minutes. Then, a transparent fraction (supernatant part) obtained by centrifuging this sample at 15,000 rpm for 20 minutes was collected.
2. Electrophoresis of protein fraction
The supernatant fraction obtained here was fractionated by SDS polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue R250, and then analyzed. The analysis results are shown in FIG. In FIG. 4, a black diamond mark indicates a D hordein band.
FIG. 4 shows, as an example, electrophoresis of a USD buffer soluble fraction of 3 T1 seeds of 90113AT15-1-4 line into which DPADH6NPT was introduced. As a negative control, commercially available Igri seeds and seeds attached to redifferentiated plants into which no gene had been introduced were used.
As shown in FIG. 4, in lanes 5 and 7, which are hordein fractions of the transformants, the relative content of D hordein relative to other hordeins compared to the non-transformed control (lanes 2, 3, 4). , It became clear that it decreased dramatically.
In addition, in other lines into which the D hordein expression suppression vector was introduced, it was revealed that the relative content of D hordein decreased compared to the control, although there were some differences. Therefore, the D hordein expression-suppressing vector constructed in the present invention has an effect of reducing D hordein specifically in transformed barley, that is, almost without changing the content of other hordeins. I can conclude.
In addition, although the difference was not recognized with the control | contrast in the lane 6, it can be explained that this result was caused because the transgene was separated in the T1 seed. Thus, this fact is not inconsistent with the above conclusion that the decrease in D hordein occurs as a result of the introduction of the D hordein expression suppression vector.
[Example 4] Determination of gel protein
1. Gel protein recovery from seeds
Gel protein was quantified with reference to Skerritt and Janes (Skerritt and Janes (1992), Journal of Cereal Science 16: 219-235).
After peeling the skin of one T1 seed obtained from the transformant and crushing it finely with a hammer, placing it in a centrifuge tube that has been weighed in advance, and drying it for 30 minutes using Speed Vac Concentrater (manufactured by Savant), The weight was measured and the weight of the kernel was calculated. To this sample, 3 ml of 1.5% (w / v) SDS was added and stirred for 1 hour by vortexing.
The sample after stirring was centrifuged at 40000 × g and 20 ° C. for 1 hour, and the resulting supernatant was designated as “SDS soluble fraction” (or “SDS fraction”).
On the other hand, the fraction precipitated in the above centrifugation was 1.5% (w / v) SDS / 10% (v / v) 2-mercaptoethanol / 16% (v / v) N, N′-dimethylformamide (hereinafter, 2 ml of SDM) was added, stirred well, and incubated at 45 ° C. for about 20 hours. The sample after this incubation was centrifuged at 40000 × g and 20 ° C. for 1 hour, and the resulting supernatant was dialyzed using 0.2% SDS. This dialysis was repeated 3 times for 2 hours, and the volume was measured after dialysis. The fraction obtained here is a “gel protein fraction” (or “gel fraction”).
2. Reduction of gel protein due to decrease of D hordein
For the protein quantification of the SDS fraction and the gel fraction, BCA Protein Assay Reagent (manufactured by Pierce) was used, and bovine serum albumin was used as a standard protein. After quantification of the gel protein of the transformant, whether or not D hordein was reduced in the T1 seed used was examined by SDS polyacrylamide electrophoresis, and the transgene was separated (dropped out). Data for T1 seeds where no reduction was observed was deleted from here.
Transformant lines (90113AT1-2C, 90113AT14-) into which a D hordein expression suppression vector was introduced, using barley cultivar Igri and redifferentiated barley lines (90213AC1-5A, 90213AC1-6B, 90213AC1-4B) without gene transfer as negative controls. The protein content of the gel fraction obtained from T1 seeds of 1-3C, 90113AT14-1-5G, 90113AT15-1-4) was examined.
In addition, extraction and quantification were performed using at least 5 samples for each line (n ≧ 5). FIG. 5A shows the amount of gel protein and the standard error in the gel fraction extracted from 1 g of sample.
As is clear from the graph shown in FIG. 5A, in the group where the D hordein content decreased (FIG. 5A: 5, 6, 7, 8), compared to the negative control (FIG. 5A: 1, 2, 3, 4), The amount of gel protein was clearly reduced. In addition, the average amount of gel protein in all samples with reduced D hordein was significantly lower than the average value of all negative control samples (approximately 40% decrease in average value). Level 1%). From the above results, it is shown that the decrease in gel protein is not caused by a culture mutation or the like, but is a phenomenon caused by a decrease in D hordein.
3. Confirmation of the relationship between decrease in D-hordein and decrease in gel protein
We examined how the amount of gel protein changes in T1 seeds grown in the same individual depending on whether or not the amount of D-hordein decreased as a result of transgene separation.
About 10 T1 seeds obtained from the same individual of the 90113AT15-1-4 line, gel protein was extracted, and the amount of D hordein decreased qualitatively by electrophoresis. , It was divided into groups (5 grains) in which no decrease was observed. Then, using these two groups of samples, the relationship between the presence or absence of this D hordein reduction and the amount of gel protein was investigated. The result is shown in FIG. 5B.
As shown in FIG. 5B, the amount of gel protein in the seed group (5 grains) in which the amount of D hordein was reduced (FIG. 5B: Sample 2) was the gel of seeds (5 grains) in which the decrease in the amount of D hordein was not observed. It became clear that it was significantly lower than the amount of protein (FIG. 5B: Sample 1) (1% significant level).
Since this is a test in which seeds grown on the same individual were examined, the difference in the growth environment that occurs between individuals can be ignored. Therefore, it is shown that the decrease in gel protein is not a result due to the difference in growth environment but a phenomenon that occurs as a characteristic of an individual, that is, a characteristic that the production amount of D-hordein protein is specifically reduced. It was.
From the above results, it can be concluded that a reduction in the amount of gel protein is made possible by introducing a D hordein expression suppression vector and reducing the amount of D hordein protein produced.
[Example 5] Compositional change of gel protein
The composition change of the gel protein produced by introducing the D hordein expression suppression vector was examined. SDS-polyacrylamide gel electrophoresis of the SDS fraction extracted from 90113AT15-1-4 (FIG. 6: lanes 2, 3, 4, 5) and the gel fraction (FIG. 6: lanes 6, 7, 8, 9), respectively. Analyzed with
As a result, in the T1 seed (FIG. 6: lanes 4 and 8) having a reduced D hordein content compared to the control (FIG. 6: lanes 2, 3, 5, 6, 7, and 9), the gel fraction was originally It was clarified that a specific protein (hereinafter referred to as “GS protein”, indicated by an arrow in FIG. 6) extracted in a large amount is transferred to the SDS fraction.
As a result, the reduction of the gel protein caused by introducing the D hordein expression suppression vector is not only due to the decrease of D hordein, but also the proteins (including at least GS protein) constituting other gel proteins. It is thought to be caused by removal from the gel fraction.
[Example 6] Transmission of progeny to progeny
It was examined whether the introduced trait described in the above example was transmitted to progeny. As a result of extracting and quantifying gel protein from 7 T2 seeds with reduced D hordein, an average of 16.9 mg (± 1.48) gel protein was extracted per gram of grain. This amount of gel protein was almost the same as that of the parent T1 seed with reduced D hordein, and it was revealed that the trait of reduced gel protein was stably transmitted to progeny.
Industrial applicability
According to the present invention, it has been clarified that barley with reduced gel protein, which is considered to be highly related to wort filterability and wort extract amount, can be easily and efficiently produced. By using the present invention, it is possible to reduce the time and labor conventionally required for the mating breeding method and the sudden breeding method, and without using the breeding field or the radiation irradiation apparatus necessary for them. It is possible to obtain barley with reduced gel protein.
[Sequence Listing]

Claims (6)

Inhibition of D hordein expression in which the nucleotide sequence of SEQ ID NO: 1 or 2 is linked in the reverse direction downstream of a promoter operating in barley so that an antisense RNA complementary to the endogenous D hordein RNA of barley can be generated Nucleic acid is introduced into barley to suppress the production of D hordein protein and reduce the amount of gel protein, thereby producing a barley with reduced gel protein that can be aggregated in a gel form during protein extraction from barley Method. The base sequence described in SEQ ID NO: 1 or 2 is ligated in the reverse direction downstream of a promoter that operates in barley so that an antisense RNA complementary to the endogenous D hordein RNA of barley can be generated. D-hordein expression-suppressing nucleic acid. A vector comprising the D hordein expression-inhibiting nucleic acid according to claim 2. 4. The vector according to claim 3, further comprising a selectable marker expression cassette capable of expressing a selectable marker in the vicinity of the D hordein expression-suppressing nucleic acid. Including the D hordein expression-suppressing nucleic acid according to claim 2 or the vector according to claim 3 or 4, wherein the D hordein expression-suppressing nucleic acid or vector is introduced into barley to reduce the barley gel protein, Barley production kit with reduced gel protein. A barley produced by the method for producing a barley protein-reduced barley according to claim 1 or the kit for producing a barley protein-reduced barley according to claim 5, and comprising the D hordein expression-suppressing nucleic acid according to claim 2.

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